The overall objective of the research proposed is to create a new RNA virus vaccine using a novel self-assembling nanoparticle packaging method, designed to overcome safety, cost and other limitations that slow vaccine development. Virus-based vaccines have the ability to stimulate both innate and adaptive immunity, which can improve vaccine potency compared to other methods of vaccine delivery. RNA virus based vaccines have improved safety compared to DNA virus vaccines because they can't integrate into the host genome, but are still limited by cell-based capsid packaging methods that are costly and limit vaccine boosting. Our preliminary data show that by removing all native capsid assembly constraints self-assembling vaccines can be made with improved safety, vaccine stability, and at very low cost, simply by mixing RNA and coat protein together. The Specific Aims of this research plan are 1) to modify the insect Flock House Virus RNA genome with a non-native capsid origin of assembly and confirm in vitro particle formation, 2) to insert a foreig reporter transgene under the control of a virus promoter and evaluate the protein accumulation in cells, and 3) to combine the transgene expression and nanoparticle self-assembly, and to confirm that both are functional. The resulting product is a viral RNA vaccine with packaging characteristics independent of its native capsid, while retaining the virus characteristics that make it a good vaccine antigen. That includes nM particulate size for optimal antigen uptake by immune cells, stability at room temperature for years, and the ability to safely carry viral transgene expression into immune cells without the risk of virus reconstitution. Flock House Virus was selected for self-assembly because it also has many desirable characteristics. It is not a mammalian pathogen, and thus will exhibit improved safety. It has very high levels of antigen expression in mammalian cell types, and greatly reduced cell death compared to RNA viruses currently used in vaccine development. Flock House Virus has not been previously exploited for vaccine development because the native capsid packaging characteristics significantly limit transgene insert size and the virus particles still need to be made in cell culture. The expected outcome of this application will be to overcome native capsid packaging limitations, and create a self-assembling RNA nanoparticle based on the Flock House Virus genome, with high level antigen expression. Our research is significant, because it will fulfill our long-term objective to create cost effective, safe and robust RNA vaccines, and it is innovative because we will validate that any RNA virus with desirable characteristics can be adapted for nanoparticle self- assembly, and increase the pace of RNA vaccine development for human use. PUBLIC HEALTH RELEVANCE: Improving vaccine characteristics by rational design is a high priority of research scientists and the medical community, because vaccines play a critical role in improving public health. This R03 application seeks to improve vaccine characteristics by testing a new type of self-assembling RNA vaccine nanoparticle, which unlike currently approved vaccines, is rapidly and easily made, is stable at room temperature, and can be made cost effectively. Our goal is to implement improved RNA based vaccines that can be used clinically, and this will have a positive impact on improved safety, superior immune activation and protection against infectious disease pathogens and cancer, and may facilitate widespread adoption of new vaccines in the future.